Project Summary/Abstract Research demonstrates that extracellular matrix (ECM) stiffness dictates the differentiation of the mesenchymal stem cells (MSCs) attached to it. Stiffer substrates promote the differentiation of stiffer cell lineages [1]. Photodegradable hydrogels are a novel class of polymeric biomaterials that have been developed by our group at University of California, Los Angeles. They are uniquely suited to replicate the ECM[2, 3] and are physically and chemically similar to the ECM, having a wide range of elastic moduli (~1-500 kPa). Distinctive of photodegradable hydrogels is that their cross-link density can be altered externally, where stiffness is a function of the light intensity and exposure time. This biomaterial can replicate complex, anisotropic, and heterogeneous microenvironments that mimic the structural heterogeneities of native tissue with sub-micron resolutions. This project proposes to advance tissue engineering through the design and use of this photodegradable hydrogel as a high-precision advanced biomaterial, with five degrees of control (three spatial dimensions, time and intrinsic property gradation). I intend to mimic patterns and stiffness gradations found in native tissue interfaces in order to analyze MSC behavior and advance the understanding of stem cell fate in vivo. The mechanical patterns I intend to create will ultimately replicate the inherently complex environments found in nature, such as the developing tooth and the bone-cartilage interface of the mandibular condyle. To date, ECM-based stem cell research has not replicated the body's polarized structures to a degree that allows for an understanding of cell fate in such environments. This research proposal intends to establish how heterogeneous mechanical environments impact MSCs in 2D and 3D culture to subsequently answer the following: Are microenvironments that contain polarized structures dictating stem cell fate? If we engineer an environment to mimic native conditions, will stem cells follow suit? Hypothesis: Photo-tunable polymer networks can be used to structure heterogeneities similar to those found native tissue by inducing a cellular response to mechanical cues. Since it has been established that cell differentiation can be triggered mechanically in static isotropic materials, a system with spatial differences in mechanical properties should trigger multiple cell lineages within a continuous material across a multitude of length and time scales. I also expect that beyond controlling cell phenotype, intermediate behavior, as found in native tissue interfaces, will be observed.